Recombinant Salmonella enteritidis PT4 Protein AaeX (aaeX)

What is the structural characterization of Salmonella enteritidis PT4 Protein AaeX?

Salmonella enteritidis PT4 Protein AaeX (aaeX) is a small membrane protein consisting of 67 amino acids with the sequence: MSLFPVIVVFGLSFPPIFFELLLS LAIFWLVRRMVPTGIYDFW WPALFNTALYCCLFYLISRLFV . The protein is encoded by the aaeX gene, identified in the Salmonella enteritidis PT4 strain P125109 genome with the UniProt accession number B5R1A9 .

For structural characterization, researchers typically employ:

• Circular dichroism spectroscopy to analyze secondary structure elements

• Hydrophobicity analysis suggesting a transmembrane topology

• Bioinformatic prediction tools to identify conserved domains

The hydrophobic nature of the N-terminal region indicates a probable membrane-anchoring domain, while the C-terminal region contains charged residues that may interact with cytoplasmic or periplasmic components.

What expression systems are most effective for recombinant production of Salmonella enteritidis PT4 Protein AaeX?

The optimal expression system for recombinant AaeX production depends on research objectives. Based on available data:

For membrane proteins like AaeX, E. coli-based expression systems require optimization of detergents for membrane extraction. Regardless of system, recombinant AaeX proteins should achieve ≥85% purity as determined by SDS-PAGE for reliable experimental use .

How can researchers verify the functionality of recombinant Salmonella enteritidis PT4 Protein AaeX?

Functional verification of recombinant AaeX requires:

• Immunological confirmation: Western blotting using anti-AaeX antibodies

• Membrane localization assays: Subcellular fractionation followed by detection in membrane fractions

• Complementation studies: Introducing recombinant AaeX into aaeX-knockout strains to restore wild-type phenotype

• Binding assays: If AaeX interacts with specific host factors

Researchers should establish appropriate positive and negative controls, including:

• Positive control: Native AaeX from S. enteritidis PT4

• Negative control: Unrelated recombinant protein of similar size

Since AaeX is conserved across multiple bacterial species including Salmonella paratyphi B, Salmonella schwarzengrund, and Escherichia coli , comparative functional analysis between homologs can provide additional verification of protein activity.

How can researchers optimize recombinant Salmonella enteritidis systems for potential therapeutic applications?

Salmonella enterica strains have demonstrated promising potential as delivery vehicles for antitumor molecules, with several key considerations for researchers optimizing such systems:

The development of recombinant Salmonella enterica strains requires strategic attenuation through mutations in specific pathways while preserving tumor-targeting capabilities. Effective attenuating mutations include:

• Metabolic pathway modifications:

  • Purine biosynthesis mutations

  • Auxotrophic mutations in aromatic amino acid synthesis genes (aroA, aroC, aroD)

• Virulence reduction modifications:

  • Lipid A structure modifications (msbB mutations)

  • Type III secretion system alterations

For therapeutic applications targeting cancer, researchers should consider that Salmonella enterica has demonstrated intrinsic antitumor activity in multiple cancer models:

• Murine cancer models (sarcomas, leukemia, colon cancer)

• Human cancer xenotransplantation models (prostate, breast, osteosarcoma)

• Patient-derived orthotopic xenograft (PDOX) models

When incorporating AaeX into recombinant Salmonella therapeutic systems, researchers should:

• Evaluate if AaeX overexpression affects bacterial tropism for tumor tissues

• Determine if AaeX can serve as a fusion partner for delivery of therapeutic proteins

• Assess whether AaeX modulates host immune responses that might enhance antitumor effects

What technical challenges exist in structural studies of Salmonella enteritidis PT4 Protein AaeX and how might they be overcome?

As a small membrane protein, AaeX presents several technical challenges for structural characterization:

An integrated structural biology approach combining:

This multi-technique strategy may overcome individual method limitations to elucidate AaeX structure-function relationships.

What are the optimal conditions for assessing AaeX protein interactions with host immune cells?

For researchers investigating AaeX interactions with host immune cells, methodological considerations include:

• Cell models selection:

  • Macrophage lines: HD11 and MQ-NCSU represent appropriate models for studying phagocyte interactions

  • Lymphocyte lines: LSCC-1104-X5, LSCC-RP9 (B-cells) and MDCC-MSB-1 (T-cells) for adaptive immunity interactions

• Experimental parameters for cell infection studies:

  Parameter	Recommendation	Justification
  Multiplicity of infection	10-50 bacteria per cell	Ensures detectable internalization without overwhelming host cells
  Infection duration	0-48 hours	Covers initial entry (0-2h) and longer survival dynamics
  Temperature	37°C for mammalian; 41°C for avian cells	Physiologically relevant
  Controls	Wild-type S. enteritidis; ΔaaeX mutant; complemented strain	Isolates AaeX-specific effects

• Quantification methods:

  • Gentamicin protection assay to distinguish intracellular from extracellular bacteria

  • Confocal microscopy with fluorescently-labeled bacteria and specific subcellular markers

  • Flow cytometry for high-throughput analysis of host-pathogen interactions

When comparing results across different cell types, researchers should normalize data to account for differences in phagocytic capacity and bacterial replication rates.

How should researchers design experiments to elucidate the function of AaeX in Salmonella virulence?

A comprehensive experimental approach to determine AaeX's role in virulence should include:

• Genetic manipulation strategies:

  • CRISPR-Cas9 for precise deletion of aaeX

  • Complementation with wild-type and mutated versions of aaeX

  • Conditional expression systems to control timing of aaeX expression

• In vitro virulence assays:

  • Adhesion and invasion assays using epithelial cell lines

  • Survival within professional phagocytes (macrophages)

  • Biofilm formation capacity

  • Resistance to antimicrobial peptides and oxidative stress

• In vivo infection models:

  • Murine typhoid fever model

  • Chicken gastrointestinal colonization model

  • Competition assays between wild-type and ΔaaeX mutants

• Transcriptomic and proteomic analyses:

  • RNA-seq to identify genes differentially expressed in ΔaaeX mutants

  • Comparative proteomics of membrane fractions

  • Phosphoproteomics to identify signaling pathways affected by AaeX

Data interpretation should consider potential compensatory mechanisms that may mask AaeX phenotypes, necessitating careful experimental design with appropriate controls and statistical power calculations.

What approaches can be used to investigate potential AaeX homologs across different Salmonella species and other enteric bacteria?

For comparative analysis of AaeX homologs across bacterial species:

• Bioinformatic approaches:

  • Position-specific scoring matrix (PSSM) searches instead of simple BLAST

  • Hidden Markov Model (HMM) profiles of AaeX family

  • Phylogenetic analysis including potential homologs from Salmonella paratyphi B, Salmonella schwarzengrund, Salmonella heidelberg, and Escherichia coli

  • Synteny analysis to identify conserved genomic contexts

• Functional complementation:

  • Express heterologous AaeX proteins in S. enteritidis ΔaaeX background

  • Quantify restoration of phenotypes (invasion, survival, etc.)

  • Create chimeric proteins from different species to map functional domains

• Structure-function relationship studies:

  • Identify conserved amino acid residues across homologs

  • Site-directed mutagenesis of these residues

  • Assess impact on protein localization and function

This comparative approach can reveal evolutionary conservation patterns that suggest functional importance of specific protein regions.

How can researchers optimize immunological detection of AaeX protein in experimental systems?

For researchers developing immunological detection methods for AaeX:

• Antibody development strategies:

  • Target unique epitopes in the C-terminal region (YCCLFYLISRLFV)

  • Consider synthetic peptide immunization rather than whole protein

  • Develop both polyclonal and monoclonal antibodies for different applications

• Western blot optimization:

  • SDS-PAGE with tricine gels optimized for small proteins

  • Transfer conditions: 100V for 1 hour using PVDF membrane (0.2μm pore size)

  • Blocking: 5% non-fat milk in TBST (minimal cross-reactivity)

  • Primary antibody dilution: Start at 1:1000 and optimize

• Immunofluorescence protocol refinements:

  • Fixation: 4% paraformaldehyde followed by membrane permeabilization

  • Primary antibody incubation: 4°C overnight

  • Secondary antibody: Fluorophore selection based on imaging system

  • Controls: Include pre-immune serum and peptide competition controls

• ELISA development considerations:

  • Coating buffer optimization for hydrophobic proteins

  • Sensitivity enhancement using amplification systems

  • Standard curve generation using purified recombinant AaeX

These methodologies should be validated using both recombinant AaeX and native protein from Salmonella enteritidis PT4, with appropriate positive and negative controls throughout.

What considerations are important when designing recombinant AaeX fusion proteins for functional studies?

When designing AaeX fusion proteins for functional studies, researchers should consider:

• Fusion orientation decisions:

  • N-terminal fusions may disrupt membrane localization

  • C-terminal fusions could interfere with potential functional domains

  • Consider dual-tagging approaches for confirmation of full-length expression

• Tag selection criteria:

  Tag Type	Advantages	Limitations	Best Applications
  His6	Small size, minimal interference	Potential nonspecific binding	Purification, not ideal for localization
  FLAG/HA	Small, highly specific antibodies available	May alter protein trafficking	Immunoprecipitation, localization
  GFP/fluorescent proteins	Direct visualization	Large size may disrupt function	Live imaging, trafficking studies
  Split reporter systems	Allows protein-protein interaction studies	Complex design, potential false positives	Interactome studies

• Linker design principles:

  • Flexible linkers (GGGGS)n for independent domain function

  • Rigid linkers (EAAAK)n to separate functional domains

  • Cleavable linkers for tag removal post-purification

• Expression vector selection:

  • Inducible promoters for controlled expression

  • Strength of promoter matched to solubility of fusion

  • Consideration of codon optimization for expression host

Researchers should validate that fusion proteins maintain the expected subcellular localization and conduct comparative functional assays between tagged and untagged versions to ensure tag addition does not significantly alter protein function.

What are the critical parameters for optimizing cell-based assays to study AaeX function in host-pathogen interactions?

For cell-based assays investigating AaeX in host-pathogen interactions:

• Cell culture optimization:

  • For macrophage lines (HD11, MQ-NCSU): Culture in appropriate media supplemented with 5-10% fetal bovine serum

  • For lymphocyte lines (LSCC-1104-X5, LSCC-RP9, MDCC-MSB-1): Specialized media formulations with growth factors

  • Cell passage number: Use cells between passages 5-15 for consistent results

  • Cell density: Seed at 5×10^5 cells/well for 24-well plates

• Infection protocol standardization:

  • Bacterial preparation: Mid-log phase cultures (OD600 0.4-0.6)

  • Washing steps: 3× PBS to remove media components that may affect infection

  • Synchronization of infection: Centrifugation at 500×g for 5 minutes

  • Duration: 1 hour for initial invasion followed by gentamicin treatment

• Critical controls:

  • Heat-killed bacteria (negative control for active invasion)

  • Inhibitors of cellular processes (cytochalasin D for actin-dependent uptake)

  • Positive control strains with known invasion capabilities

  • Mock-infected cells for baseline cellular responses

• Quantification methods standardization:

  • CFU counting: Plating serial dilutions on selective media

  • Microscopy: Minimum of 100 cells counted across multiple fields

  • Flow cytometry: Consistent gating strategy for infected vs. uninfected cells

Based on previous studies with Salmonella enteritidis PT4, researchers should note that macrophages typically show higher initial bacterial uptake but better clearance over 48 hours compared to lymphocyte lines . This pattern may be informative when interpreting AaeX-specific effects.

How might AaeX contribute to Salmonella enteritidis adaptation during different phases of infection?

Understanding AaeX's role in different infection phases requires:

• Temporal expression analysis:

  • qRT-PCR analysis of aaeX expression during:

    • Early attachment and invasion (0-2 hours)

    • Intracellular survival phase (2-24 hours)

    • Persistent infection (24-72 hours)

  • Promoter-reporter fusions to visualize expression in real-time

• Environmental regulation studies:

  • Expression response to pH changes (gastric to intestinal transition)

  • Nutrient limitation effects (iron, carbon sources)

  • Oxidative and nitrosative stress conditions

  • Host antimicrobial peptide exposure

• Tissue-specific function analysis:

  • Intestinal epithelial models

  • Macrophage infection models

  • Gallbladder colonization models

  • Systemic infection models

Since Salmonella enteritidis PT4 shows differential survival patterns in different host cell types , researchers should investigate whether AaeX expression correlates with bacterial persistence in specific cellular niches, potentially indicating adaptation functions for particular host environments.

Could AaeX serve as a target for developing novel antimicrobial strategies against Salmonella infections?

To evaluate AaeX as a potential antimicrobial target:

• Target validation criteria:

  • Essentiality: Determine if aaeX is essential for virulence or survival

  • Conservation: Assess presence and conservation across Salmonella strains

  • Accessibility: Confirm membrane orientation for potential drug binding

  • Absence in host: Verify no significant homology to human proteins

• Small molecule screening approaches:

  • In silico docking studies against predicted structure

  • High-throughput screening using bacterial growth inhibition

  • Targeted library screening of membrane protein inhibitors

  • Phenotypic screening for virulence attenuation

• Alternative targeting strategies:

  • Immunological targeting with anti-AaeX antibodies

  • Peptide inhibitors designed to mimic interaction partners

  • Antisense oligonucleotides to reduce expression

  • CRISPR-Cas delivery systems targeting aaeX

• Resistance development assessment:

  • Serial passage experiments with sub-inhibitory concentrations

  • Whole genome sequencing of resistant variants

  • Fitness cost analysis of resistance mutations

The presence of AaeX homologs across multiple bacterial species suggests evolutionary conservation that might indicate functional importance, potentially making it a valuable target for broad-spectrum approaches.

What is the potential for using structure-function analysis of AaeX to enhance Salmonella-based therapeutic delivery systems?

Structure-function insights into AaeX could enhance therapeutic applications through:

• Engineering principles for improved vaccine vectors:

  • If AaeX affects membrane properties, modifications could alter antigen presentation

  • Structure-guided mutations might modulate immunogenicity

  • Fusion with antigenic epitopes at permissive sites identified through structural analysis

• Optimization for cancer therapeutics delivery:

  • Given Salmonella enterica's demonstrated tumor-targeting abilities , AaeX modifications could:

    • Enhance bacterial persistence in tumor microenvironment

    • Improve secretion of therapeutic payloads

    • Modulate host immune response within tumors

• Rational design approaches:

  • Computational modeling to predict effects of mutations

  • Alanine scanning mutagenesis to identify critical residues

  • Domain swapping with homologs to create chimeric proteins with novel properties

• Expression system refinements:

  • Similar to optimization strategies used for recombinant adeno-associated virus production , structural insights into AaeX could inform:

    • Promoter design for optimal expression timing

    • Codon optimization strategies

    • Post-translational modification requirements

Research in this direction could benefit from techniques developed for other recombinant protein expression systems, such as the independently controllable expression elements used in viral vector production systems , adapted for bacterial expression contexts.